APLN-targeted antisense oligonucleotide and application thereof

Abstract

The invention provides APLN-targeted antisense oligonucleotide and application thereof, and relates to the technical field of biology. The nucleotide sequence of the APLN-targeted antisense oligonucleotide comprises a sequence, wherein the difference between the sequence of the APLN-targeted antisense oligonucleotide and the sequence shown in any one of SEQ ID NO.1-63 is not more than 1, 2, 3, 4, 5, 6 or 7 nucleotides. The antisense oligonucleotide can degrade APLN mRNA and reduce the blood vessel length and blood vessel node density of a subject, and can be used for preparing drugs for treating APLN related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and in particular to an antisense oligonucleotide targeting APLN and application thereof. BACKGROUND

[0002] The following statements are only provided with the background information related to the present application, and do not necessarily constitute the prior art.

[0003] Apelin (APLN) is a small secreted protein, which is first isolated from bovine stomach extract and later identified as an endogenous ligand for the Apelin receptor (APLNR, also known as APJ). The Apelin / APJ system is widely distributed in the body and is highly expressed in many vascular systems, such as the cardiovascular system, pulmonary vascular system, etc. Studies have found that Apelin can regulate vascular tone, promote vascular smooth muscle cell proliferation, retinal neovascularization, and monocyte adhesion to endothelial cells, promote hepatic portal vein and coronary artery collateral formation, etc., and is closely related to diseases such as hypertension, pulmonary hypertension, atherosclerosis, peripheral arterial disease, glioma, lung cancer, hepatocellular carcinoma, portal hypertension, type 2 diabetes, diabetic vascular complications, obesity, retinopathy of prematurity, diabetic retinopathy, central retinal vein occlusion, age-related macular degeneration, and irreversible visual loss, etc., and can be used as a therapeutic target for a variety of diseases. Apelin is also crucial to eye health, and the Apelin / APJ system induces various eye diseases such as retinopathy of prematurity, diabetic retinopathy, central retinal vein occlusion, age-related macular degeneration, and irreversible visual loss by stimulating pathological retinal angiogenesis.

[0004] Antisense oligonucleotides (ASO) is a synthetic DNA fragment that can bind to target mRNAs of specific sequences, thereby inducing them to be cut into fragments, achieving the effect of inducing gene silencing. Therefore, it is currently in great need to develop an antisense oligonucleotide that can effectively target APLN to inhibit the activity of APLN in a subject.

[0005] In view of the above, the present application is proposed. SUMMARY

[0006] The present application aims to provide an antisense oligonucleotide targeting APLN to achieve inhibition or blocking of APLN in a subject.

[0007] To solve the above technical problems, the present application adopts the following technical solutions:

[0008] In a first aspect, there is provided an antisense oligonucleotide targeting APLN, wherein the nucleotide sequence of the antisense oligonucleotide comprises a sequence differing by no more than 1, 2, 3, 4, 5, 6, or 7 nucleotides from any one of SEQ ID NO. 1-63.

[0009] In a second aspect, there is provided a complex comprising: (a) the antisense oligonucleotide or salt thereof of the first aspect; and (b) one or more delivery vehicles linked to (a).

[0010] In a third aspect, there is provided the antisense oligonucleotide targeting APLN or salt thereof of the first aspect, or the complex of the second aspect for use in any one of:

[0011] (I) reducing the level of APLN mRNA in a subject;

[0012] (II) preparing a medicament for reducing the level of APLN mRNA in a subject;

[0013] (III) reducing the length of blood vessels and / or the density of vascular nodes in a subject;

[0014] (IV) preparing a medicament for reducing the length of blood vessels and / or the density of vascular nodes in a subject;

[0015] (V) preparing a medicament for treating, preventing, and / or alleviating a pathological condition or disease caused by APLN in a subject;

[0016] (VI) treating, preventing, and / or alleviating a pathological condition or disease caused by APLN in a subject.

[0017] In a fourth aspect, there is provided a pharmaceutical composition comprising the antisense oligonucleotide targeting APLN or salt thereof of the first aspect, or the complex of the second aspect.

[0018] In a fifth aspect, there is provided a method of inhibiting APLN in a subject, the method comprising contacting the subject with the antisense oligonucleotide targeting APLN or salt thereof of the first aspect, or the complex of the second aspect, or the pharmaceutical composition of the fourth aspect.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] The present application finds that the degradation of APLN mRNA and / or the reduction of the length of blood vessels and the density of vascular nodes in a subject can be achieved by ASO of specific sequences. Through screening, ASO with high knockdown efficiency is obtained, which can be used for preparing a medicament for treating and / or preventing a disease related to APLN. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the description of the embodiments or the prior art. Obviously, the drawings described below are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort based on these drawings.

[0022] Figure 1 Average vessel length results for antisense oligonucleotides of different sequences in Example 3 in the in vitro angiogenesis assay;

[0023] Figure 2 Node density results for antisense oligonucleotides of different sequences in Example 3 in the in vitro angiogenesis assay;

[0024] Figure 3 Average vessel length results for antisense oligonucleotides of different sequences in Example 4 after free diffusion to HUVEC;

[0025] Figure 4 Node density results for antisense oligonucleotides of different sequences in Example 4 after free diffusion to HUVEC;

[0026] Figure 5 Average vessel length results for MsPA modified antisense oligonucleotides in Example 7 after free diffusion to HUVEC;

[0027] Figure 6 Node density results for MsPA modified antisense oligonucleotides in Example 7 after free diffusion to HUVEC. DETAILED DESCRIPTION

[0028] The technical solutions of the present application will be described below in conjunction with the embodiments, obviously, the described embodiments are some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative effort belong to the scope of protection of the present application.

[0029] In this document, the term "comprising" or "including" is an open-ended expression, which is used in the present disclosure to mean "including but not limited to", and is used interchangeably with "consisting of.

[0030] In this document, the term "optionally", "optional" or "optional" generally means that the event or condition described subsequently can but does not necessarily occur, and the description includes both cases where the event or condition occurs and cases where the event or condition does not occur.

[0031] As used herein, "and / or" means one or the other or both, for example, A and / or B includes (A and B) and (A or B).

[0032] As used herein, unless otherwise indicated, any number is used to distinguish one entity or action from another, and does not necessarily imply any actual such relationship, order, or importance of the entities or actions, for example, i, ii; first, second, etc.

[0033] As used herein, the term "comprising" or "including" means including the recited elements, integers or steps, but not excluding any other elements, integers or steps.

[0034] As used herein, an antisense oligonucleotide (ASO) refers to a short chain of nucleic acid fragments that can bind complementarily to a target nucleic acid. As used herein, an antisense oligonucleotide includes ribonucleotides and / or deoxyribonucleotides, including but not limited to DNA or RNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. As used herein, unless otherwise indicated, a nucleotide refers to a ribonucleotide and / or a deoxyribonucleotide. An antisense oligonucleotide can specifically hybridize to a target nucleic acid or a region or segment thereof, and the hybridization results in RNase H-mediated cleavage of the target nucleic acid. In alternative embodiments, an antisense oligonucleotide targeting APLN includes targeting APLN mRNA or pre-mRNA.

[0035] As used herein, a "spacer" or "gapmer" means an antisense oligonucleotide having the following characteristics, comprising an internal region of a plurality of nucleosides that support RNase H cleavage positioned between external regions of one or more nucleosides, wherein the nucleosides comprising the internal region are chemically different from the one or more nucleosides comprising the external regions. The internal region can be referred to as a "gap segment" or "spacer segment", and the external regions are referred to as "wing segments". In alternative embodiments, the antisense oligonucleotide is a gapmer.

[0036] The term "complementary" is used to describe the relationship between nucleotide bases and / or polynucleotides that are capable of hybridizing to one another, e.g., the nucleotide sequence of such a polynucleotide or region(s) thereof matches the nucleotide sequence of another polynucleotide or region(s) thereof when the two nucleotide sequences are aligned in opposite directions. As described herein, nucleobase matches or complementary nucleobases include the following pairs: adenine (A) with thymine (T), adenine (A) with uracil (U), cytosine (C) with guanine (G), and 5-methylcytosine (mC) with guanine (G). Complementary polynucleotides and / or nucleic acids need not have nucleobase complementarity at every nucleoside and can include one or more nucleobase mismatches. Accordingly, the present disclosure also includes isolated polynucleotides complementary to sequences as disclosed or used herein, as well as those substantially similar nucleic acid sequences. The extent to which two polynucleotides have matching nucleobases can be expressed in terms of a "percent complementarity" or "percent complement." Unless otherwise specified, the percent complement is the percent of nucleobases of a shorter sequence that are complementary to a longer sequence.

[0037] "mismatch" or "non-complementary" means that a nucleobase of a first polynucleotide is not complementary to a corresponding nucleobase of a second polynucleotide or target nucleic acid when the first and second polynucleotides are aligned. For example, a nucleobase (including, but not limited to, a universal nucleobase, inosine, and hypoxanthine) is capable of hybridizing to at least one nucleobase, but is still a mismatch or non-complementary with respect to the nucleobase to which it hybridizes. As another example, a nucleobase of a first polynucleotide that is not capable of hybridizing to a corresponding nucleobase of a second polynucleotide or target nucleic acid is a mismatch or non-complementary nucleobase when the first and second polynucleotides are aligned.

[0038] Nucleobases can be naturally occurring or synthetic. Nucleobases and sugar bases can each independently be modified or unmodified. "Modified nucleoside" means a nucleoside comprising a modified nucleobase and / or a modified sugar moiety. Modified nucleosides can include abasic nucleosides lacking a nucleobase. For ease of expression, "modified nucleoside" can be denoted herein using the unmodified nucleoside abbreviation, i.e., adenine (A), thymine (T), uracil (U), cytosine (C), guanine (G), but with the caveat.

[0039] "2'-0-methoxyethyl" (2'-MOE) refers to 2'-0(CH2)2-0CH3in place of the 2'-OH group of a ribosyl ring.

[0040] "5-methylcytosine nucleotide" means a cytosine nucleotide having a methyl group attached to the 5 position.

[0041] Herein, "contiguous" in the context of oligonucleotides refers to nucleosides, nucleobases, sugar moieties, or internucleosidic linkages that are immediately adjacent to one another. For example, "contiguous nucleobases" means nucleobases that are immediately adjacent to one another in a sequence.

[0042] Herein, "internucleosidic linkage" is the linkage between adjacent nucleotides in a polynucleotide. As used herein, "modified internucleosidic linkage" means any internucleosidic linkage other than a phosphodiester internucleosidic linkage.

[0043] Herein, "phosphorothioate internucleosidic linkage" refers to a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced by a sulfur atom.

[0044] Herein, "methionine-amino phosphonate internucleosidic linkage (MsPA)" refers to a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced by a methionine-amino group, which has the structure:

[0045]

[0046] Herein, when "linked" refers to the connection of two molecules, the two molecules can be directly linked (e.g., directly linked by a chemical bond), or linked through an additional molecule; when the two molecules are directly linked, the two molecules can be covalently or non-covalently linked. Covalently linked refers to the connection of two molecules by the formation of a covalent bond, which is a chemical bond that forms between atoms by the sharing of electron pairs; non-covalently linked refers to a connection other than covalent linkage, such as by hydrogen bonding, van der Waals forces, electrostatic interactions, hydrophobic interactions, and metal coordination bonds, among others.

[0047] Herein, the term "subject" or "patient" refers to a mammalian subject or patient, as well as organs, tissues, or cells derived therefrom. The mammal includes, but is not limited to, a human, a rhesus monkey, a rat, a mouse, a guinea pig, a rabbit, a dog, a cat, a hamster, a nude mouse, a ferret, a pig, a sheep, a goat, a cow, a horse, a donkey, a canine, a cynomolgus monkey, a rhesus monkey, a baboon, a gibbon, a golden monkey, a langur, a marmoset, a squirrel monkey, a pig-tailed macaque, or a chimpanzee.

[0048] Herein, the terms "treat," "treatment," "treatments," "treating," "amelioration," or "ameliorating" are used interchangeably. These terms refer to methods of obtaining beneficial or desired results, including but not limited to therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of one or more symptoms associated with the underlying disorder being treated.

[0049] As used herein, the terms "prevent" and "preventing" are used interchangeably, and refer to methods of obtaining beneficial or desired results, including but not limited to, preventative benefits. To obtain "preventative benefits," a drug can be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease can not have been made.

[0050] As used herein, the term "inhibit," can be used interchangeably with "reduce," "silence," "down-regulate," "repress," and other similar terms, and includes inhibition at any level. Inhibition can be assessed by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as a pre-dose baseline level or a level determined from a subject, cell, or sample that has never been treated or treated with a control, such as a buffer control or inert agent control.

[0051] In a first aspect, an antisense oligonucleotide targeting APLN is provided, wherein the nucleotide sequence of the antisense oligonucleotide comprises a sequence differing no more than 1, 2, 3, 4, 5, 6, or 7 nucleotides from any one of the sequences set forth in SEQ ID NO. 1-63.

[0052] In an optional embodiment, the difference nucleotides in the antisense oligonucleotide from the sequences set forth in SEQ ID NO. 1-63 are from mutation at any one or more positions in the sequences set forth in SEQ ID NO. 1-63, and / or from 1, 2, 3, 4, 5, 6, or 7 nucleotides adjacent to the 5' end and / or 3' end of the fragment corresponding to APLN mRNA in any one of the sequences set forth in SEQ ID NO. 1-63.

[0053] In an optional embodiment, the nucleotide sequence of the antisense oligonucleotide is any one of the sequences set forth in SEQ ID NO. 1-63.

[0054] In an optional embodiment, the antisense oligonucleotide comprises at least one modified nucleotide.

[0055] In optional embodiments, the modified nucleotides include, but are not limited to, one or more of 2'-deoxy-modified nucleotides, 2'-0-methoxyethyl modified nucleotides, 5'-methyl modified nucleotides, 2'-0-methyl modified nucleotides, 2'-fluoro modified nucleotides, locked nucleotides, non-locked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-0-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxyl-modified nucleotides, 2'-0-alkyl-modified nucleotides, morpholino nucleotides, bridged nucleic acids, peptide nucleic acids, locked nucleic acids, non-locked nucleic acids, and 2'-(S)-constrained ethyl modified nucleotides.

[0056] In optional embodiments, at least one internucleoside linkage is a modified internucleoside linkage.

[0057] In optional embodiments, the modified internucleoside linkage includes at least one of a phosphorothioate internucleoside linkage and a methylphosphonamidate internucleoside linkage.

[0058] In optional embodiments, all internucleoside linkages of the antisense oligonucleotide are phosphorothioate internucleoside linkages.

[0059] In optional embodiments, the antisense oligonucleotide contains 1, 2, or 3 methylphosphonamidate internucleoside linkages, and the remaining internucleoside linkages are phosphorothioate internucleoside linkages.

[0060] In optional embodiments, the antisense oligonucleotide is a gapmer.

[0061] In optional embodiments, the antisense oligonucleotide comprises a gap segment consisting of at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous deoxynucleotides, and a 5' wing segment and a 3' wing segment each consisting of contiguous nucleotides, wherein the gap is positioned between the 5' wing segment and the 3' wing segment.

[0062] In optional embodiments, the nucleotides in the 5' wing segment and the 3' wing segment are each modified nucleotides.

[0063] In optional embodiments, each modified nucleotide in the 5' wing segment and the 3' wing segment is independently selected from a 2'-0-methoxyethyl modified nucleotide, a 2'-methoxy modified nucleotide, a locked nucleic acid, or a bridged nucleic acid.

[0064] In optional embodiments, the nucleotides in the 5' wing segment and the 3' wing segment are each 2'-0-methoxyethyl modified nucleotides.

[0065] In an alternative embodiment, the cytosine nucleotides in the antisense oligonucleotide are all 5'-methylcytosine nucleotides.

[0066] In an alternative embodiment, the antisense oligonucleotide comprises a gap segment consisting of 10 contiguous deoxynucleotides, and a 5' wing segment and a 3' wing segment each consisting of 5 contiguous nucleotides, the nucleotides in the 5' wing segment and the 3' wing segment are all 2'-0-methoxyethyl modified nucleotides, and the cytosine nucleotides in the antisense oligonucleotide are all 5'-methylcytosine nucleotides; the antisense oligonucleotide contains 2 or 3 consecutive methoxy- aminophosphoramidate internucleoside linkages, which are located in the gap segment, and all other internucleoside linkages are phosphorothioate internucleoside linkages. In a further alternative embodiment, the nucleotide sequence of the antisense oligonucleotide is set forth in SEQ ID NO. 35.

[0067] In a second aspect, there is provided a complex comprising: (a) the antisense oligonucleotide or salt thereof according to the first aspect; and (b) one or more delivery carriers linked to (a).

[0068] In an alternative embodiment, the delivery carriers include, but are not limited to, targeted delivery carriers, which include, but are not limited to, aptamers, targeting peptides or compound drugs.

[0069] In a third aspect, there is provided a complex of an antisense oligonucleotide targeting APLN or a salt thereof, or use of the complex according to the second aspect in any one of:

[0070] (I) reducing the level of APLN mRNA in a subject;

[0071] (II) preparing a medicament for reducing the level of APLN mRNA in a subject;

[0072] (III) reducing the length of blood vessels and / or the density of vascular nodes in a subject;

[0073] (IV) preparing a medicament for reducing the length of blood vessels and / or the density of vascular nodes in a subject;

[0074] (V) preparing a medicament for treating, preventing and / or alleviating a pathological condition or disease caused by APLN in a subject;

[0075] (VI) treating, preventing and / or alleviating a pathological condition or disease caused by APLN in a subject.

[0076] In optional embodiments, the pathological condition or disease caused by APLN in the subject includes, but is not limited to, at least one of hypertension, pulmonary arterial hypertension, atherosclerosis, peripheral arterial disease, glioma, lung cancer, hepatocellular carcinoma, portal hypertension, type 2 diabetes, diabetic vascular complications, obesity, retinopathy of prematurity, diabetic retinopathy, central retinal vein occlusion, age-related macular degeneration, and irreversible vision loss.

[0077] In optional embodiments, the use of the (I) or (III) aspect is for non-diagnostic and therapeutic purposes.

[0078] In a fourth aspect, there is provided a pharmaceutical composition comprising the APLN-targeting antisense oligonucleotide or salt thereof of the first aspect, or the complex of the second aspect.

[0079] In optional embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable optional excipient. The acceptable excipient can be selected from any excipient known in the art and conventionally used. Examples of excipients include, but are not limited to, any physiologically compatible solvents, dispersion media, coatings, antibacterial agents, antifungal agents, pH adjusting agents, lyophilization protecting agents, and emulsifiers, etc.

[0080] In optional embodiments, the pharmaceutical composition comprises a delivery system for delivering the APLN-targeting antisense oligonucleotide or for delivering the complex. The delivery system includes, but is not limited to, a lipid nanoparticle, a liposome, a nanoparticle, a cationic lipid, a cationic polymer, a metal nanopolymer, a nanorod, a micelle, a microvesicle, a cell-penetrating peptide, a viral particle, a protein coat, or a liposphere.

[0081] In a fifth aspect, there is provided a method of inhibiting APLN in a subject, the method comprising contacting the subject with the APLN-targeting antisense oligonucleotide or salt thereof of the first aspect, or the complex of the second aspect, or the pharmaceutical composition of the fourth aspect.

[0082] In optional embodiments, the subject is a cell.

[0083] In optional embodiments, the subject is a human dermal fibroblast (HDF) cell or a human umbilical vein endothelial cell (HUVEC)

[0084] In optional embodiments, the subject is a cell, and the contacting comprises delivering the APLN-targeting antisense oligonucleotide or the complex into the cell using any method known in the art.

[0085] In optional embodiments, the subject is a cell and the working concentration of the antisense oligonucleotide targeting TSLP is at least 1 nM, at least 3 nM, at least 5 nM, at least 10 nM, at least 20 nM, at least 30 nM, at least 40 nM, at least 50 nM, at least 60 nM, at least 70 nM, at least 80 nM, at least 90 nM, or at least 100 nM.

[0086] In optional embodiments, the subject is an individual organism and the contacting comprises administering to the subject by any suitable route known in the art, including but not limited to, oral administration, intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, ocular administration, or rectal administration.

[0087] In optional embodiments, the method of inhibiting APLN in a subject is not for diagnostic or therapeutic purposes.

[0088] The present application is further illustrated by the following specific examples, which are not to be construed as limiting the application in any way.

[0089] Example 1 General procedure for the preparation of MOE-Gap antisense oligonucleotides by solid phase techniques

[0090] Unless otherwise indicated, all reagents and solutions used in the synthesis of the oligomeric compounds in this application were purchased from commercial sources. Standard phosphoramidite building blocks and solid supports were used to incorporate nucleoside residues including, for example, T, A, G, and m C (5'-methylcytosine nucleotide) residues. All monomer (beta-D-2'-deoxyribonucleoside and beta-D-2'-(MOE) ribonucleoside) phosphoramidite solutions used were 0.06 M in anhydrous acetonitrile.

[0091] A 500 nmol synthesis column was packed on a LK-48E synthesizer with Universal CPG solid support and the specified sequence was synthesized using the phosphoramidite coupling method. For the coupling steps, the phosphoramidite monomers were delivered in 4-fold excess of the loading on the solid support and phosphoramidite condensation was performed for 10 min. All other steps were performed according to the standard protocol supplied by the manufacturer. A 3% solution of trichloroacetic acid in dichloromethane was used to remove the dimethoxytrityl (DMT) group from the 5'-hydroxyl group of the nucleotides. 5-benzylthio tetrazole (BTT, 0.35 M in anhydrous acetonitrile containing 0.5% N-methylimidazole (NMI)) was used as the activator during the coupling steps. The phosphorothioate linkages were introduced by a 3 min contact time with a 0.2 M solution of dithiobisphenacyl (PADS) in 1:1 pyridine / acetonitrile.

[0092] After the synthesis of the specified sequence, the solid support-bound specified sequence was suspended in aqueous ammonia (25-30 wt%) and heated at 85°C for 2 h, then the solid phase support was filtered off and the ammonia was removed under reduced pressure. The residue was purified by high pressure liquid chromatography to produce the Gap antisense oligonucleotides as shown in Table 1.

[0093] Table 1

[0094]

[0095]

[0096] Note: The Gap antisense oligonucleotides in Table 1 are all 20 nucleotides in length and are designed as 5-10-5 gapmers, i.e. from 5' to 3' end, positions 1-5 are 5' wing segment, positions 6-15 are gap segment, and positions 16-20 are 3' wing segment, the gap segment contains 10 2'-deoxynucleotides and is flanked on both sides (in 5' and 3' direction) by a wing segment containing 5 nucleotides each, each nucleotide in the 5' wing segment and each nucleotide in the 3' wing segment is a 2'-MOE modified nucleotide, each nucleotide in the gap is a 2'-deoxy sugar modified nucleotide, all internucleoside linkages in the gapmer are phosphorothioate (P=S) linkages, and all cytosine residues in the gapmer are 5'-methylcytosine.

[0097] Example 2 Real-time fluorescent quantitative PCR detection of the effect of antisense oligonucleotides of different sequences on reducing APLN gene expression

[0098] The cells used in this example were human dermal fibroblast (HDF) cells (Promocell, CP-H103). The cells were seeded in 24-well plates at 1.5 x 10 5cells, 12 hours later, different sequence oligonucleotides were transfected into the corresponding wells by Lipofectamine RNAiMax (Thermo Fisher) transfection reagent, and the final concentration of oligonucleotides was 100 nM, and DEPC water was transfected into the blank control group. After 24 hours of continuous culture at 37°C and 5% CO2, the cells were treated with Trizol (Invitrogen) to extract RNA, and the obtained RNA was used as a template to obtain cDNA by using mmlv reverse transcriptase (Promega). Then, the expression of APLN gene was detected by using real-time fluorescent quantitative PCR with cDNA as a template and ACTB as an internal reference gene, and 2 -ΔΔCT Method was used for data analysis. Compared with the NC group, the knockdown efficiency of different concentrations of oligonucleotides was calculated, and the results are shown in Tables 2 and 3. In this example, a total of 54 antisense oligonucleotides were included, of which 46 oligonucleotides significantly inhibited the expression of APLN mRNA.

[0099] Table 2

[0100] No. Knockdown efficiency (%) No. Knockdown efficiency (%) Untreated -0.94 SG21-19 53.74 SG21-1 72.31 SG21-20 27.63 SG21-2 70.96 SG21-21 30.85 SG21-3 17.07 SG21-22 -65.6 SG21-4 39.08 SG21-23 46.28 SG21-5 -2.27 SG21-29 -28.46 SG21-6 52.82 SG21-33 70.92 SG21-7 70.04 SG21-34 63.68 SG21-8 47.54 SG21-35 48.73 SG21-9 41.2 SG21-36 67.13 SG21-10 60.3 SG21-40 83.74 SG21-11 75.99 SG21-41 64.03 SG21-12 67.11 SG21-42 64.67 SG21-13 -7.88 SG21-43 43.25 SG21-14 73.75 SG21-44 40.09 SG21-15 -127.02 SG21-45 59.58 SG21-16 60.94 SG21-46 20.22 SG21-17 53.75 SG21-47 40.23 SG21-18 -118.19 SG21-48 13.13

[0101] Table 3

[0102] No. Knockdown efficiency (%) No. Knockdown efficiency (%) Untreated -1 SG21-58 90.32 SG21-40 70.75 SG21-59 55.76 SG21-49 71.03 SG21-60 40.38 SG21-50 83.93 SG21-61 -16.48 SG21-51 73.31 SG21-62 12.25 SG21-52 34.92 SG21-63 61.52 SG21-53 94.4 SG21-64 79.44 SG21-54 93.53 SG21-65 -75.98 SG21-55 81.09 SG21-66 51.59 SG21-56 22.18 SG21-67 64.39 SG21-57 57.65 SG21-68 87.61

[0103] Example 3 In vitro angiogenesis experiment for detecting the inhibitory effect of different sequences of antisense oligonucleotides on angiogenesis

[0104] The cells used in this example were human umbilical vein endothelial cells (HUVEC) (Pronova, CP-H082). In a 96-well plate, 50 μL of pre-cooled Matrigel was added to each well to avoid air bubbles, and the plate was placed in a 37°C incubator for 30 minutes. The HUVEC cells were digested and counted, and 1.5 x 10 4 Lipofectamine RNAiMax transfection reagent was mixed with different sequence oligonucleotides, and then the counted HUVEC cells were added to make the final concentration of oligonucleotides 100 nM. After mixing, the mixture was added to the 96-well plate with Matrigel. After 18-24 hours, 1 ml of 1 mM calcein AM was added to 50 ml of serum-free medium to make the final concentration 20 nM. After incubation at room temperature for 30 minutes in the dark, the cells were washed with PBS for 2-3 times, and then immunofluorescence imaging was performed using 485 nm / 529 nm. AngioTool software was used for image analysis and statistics. The results are shown in Table 3. Figure 1 、 Figure 2As shown in Table 4, the average vessels length and junction density were measured. Among the 43 antisense oligonucleotides, 42 of them significantly reduced the average vessels length and 43 of them significantly reduced the junction density compared to the negative control (NC) PBS. SG 21-40 was the best in both aspects.

[0105] Table 4

[0106]

[0107]

[0108] Example 4 Inhibition of angiogenesis in vitro by antisense oligonucleotides of different sequences after free uptake into umbilical vein endothelial cells (HUVEC)

[0109] In each well of a 96-well plate, 50 μL Matrigel was added to avoid air bubbles and incubated in a 37°C incubator for 30 minutes. HUVEC cells were trypsinized and counted so that 1.5 x 10 4 The counted HUVEC cells were mixed with different concentrations of oligonucleotides so that the final concentrations were 0 mM, 1 mM, 5 mM and 10 mM, respectively. After thorough mixing, the mixture was added to the 96-well plate with Matrigel. After 18-24 hours, 1 mL of 1 mM calcein AM was added to 50 mL of serum-free medium so that the final concentration was 20 nM. After incubation at room temperature for 30 minutes in the dark, the plate was washed with PBS for 2-3 times. Immunofluorescence imaging was performed using 485 nm / 529 nm. The pictures were analyzed and counted using AngioTool software. The results are shown in Table 5. Figure 3 and Figure 4 As shown in Table 5, almost all of the tested oligonucleotides at 5 mM and 10 mM could inhibit angiogenesis to different degrees compared to the negative control (PBS) (0 mM, 100%). SG 21-40 was the most effective.

[0110] Table 5

[0111]

[0112] Example 5 General method for preparing MsPA modified antisense oligonucleotides by solid phase technique

[0113] All reagents and solutions used for the synthesis of oligomeric compounds were purchased from commercial sources unless otherwise noted. Standard phosphoramidite building blocks and solid supports were used to incorporate nucleoside residues including, for example, T, A, G, and m C residues. All monomer (beta-D-2'-deoxyribonucleoside and beta-D-2'-(MOE) ribonucleoside) phosphoramidite solutions used were 0.06 M in anhydrous acetonitrile.

[0114] A 500 nmol synthesis column was packed on an LK-48E synthesizer using a Universal CPG solid support and the indicated sequence synthesis was performed using phosphoramidite coupling methodology. For the coupling steps, phosphoramidite monomers were delivered in 4-fold excess of the loading on the solid support and phosphoramidite condensation was continued for 10 min. All other steps were performed according to the standard protocols supplied by the manufacturer. A 3% solution of trichloroacetic acid in dichloromethane was used to remove the dimethoxytrityl (DMT) group from the 5'-hydroxyl of the nucleotides. BTT (0.35 M, 0.5% NMI in anhydrous acetonitrile) was used as the activator during the coupling steps. Phosphorothioate linkages were introduced by a 3 min contact time with a 0.2 M solution of phenylacetyl disulfide (PADS) in 1 : 1 pyridine / acetonitrile. Methanesulfonyl-aminophosphonate (MsPA) linkages were introduced by a 2 min contact time with a 1 M solution of methanesulfonyl azide (MsN3) in acetonitrile.

[0115] After the synthesis of the indicated sequence, the solid support-bound indicated sequence was suspended in aqueous ammonia (25-30 wt%) and heated at 85 °C for 2 h. The solid phase support was then filtered off and the ammonia was removed under reduced pressure. The residue was purified by high pressure liquid chromatography to produce the MsPA modified antisense oligonucleotides as shown in Table 6.

[0116] Table 6

[0117] Sequence SG-339 GGGAAG u C u GGCATCAGGGACC]]> SG-340 GGGAAGC u G u GCATCAGGGACC]]> SG-341 GGGAAGCG u G u CATCAGGGACC]]> SG-342 GGGAAGCGG u C u ATCAGGGACC]]> SG-343 GGGAAGCGGC u A u TCAGGGACC]]> SG-344 GGGAAGCGGCA u T u CAGGGACC <!-- 11 -->]]> SG-345 GGGAAGCGGCAT u C u AGGGACC]]> SG-346 GGGAAGCGGCATC u A u GGGACC]]> SG-347 GGGAAG u C u G u GCATCAGGGACC]]> SG-348 GGGAAGC u G u G u CATCAGGGACC]]> SG-349 GGGAAGCG u G u C u ATCAGGGACC]]> SG-350 GGGAAGCGG u C u A u TCAGGGACC]]> SG-351 GGGAAGCGGC u A u T u CAGGGACC]]> SG-352 GGGAAGCGGCA u T u C u AGGGACC]]> SG-353 GGGAAGCGGCAT u C u A u GGGACC]]>

[0118] The "u" in the MsPA modified antisense oligonucleotides in Table 6 indicates that the linkage between adjacent nucleosides uses a methanesulfonyl-aminophosphonate (MsPA) instead of a phosphorothioate (P=S) linkage, otherwise the modifications are as in Example 1.

[0119] Example 6 Real-time fluorescent quantitative PCR to detect the effect of MsPA modified antisense oligonucleotides on reducing APLN gene expression

[0120] The cells used in this example were human dermal fibroblast (HDF) cells (Promocell, CP-H103). The cells were seeded in 24-well plates at 1.5 x 105cells per well and allowed to grow to 70-80% confluency. 5cells, 12 hours later, different sequence oligonucleotides were transfected into the corresponding wells by Lipofectamine RNAiMax (Thermo Fisher) transfection reagent, and the final concentration of oligonucleotides was 50 nM, and DEPC water was transfected into the blank control group. After 24 hours of continuous culture at 37°C and 5% CO2, the cells were treated with Trizol (Invitrogen) to extract RNA, and the obtained RNA was used as a template to obtain cDNA by using mmlv reverse transcriptase (Promega). Then, the expression of APLN gene was detected by using real-time fluorescent quantitative PCR with cDNA as a template and ACTB as an internal reference gene, and 2 -ΔΔCT Method was used for data analysis. Compared with the NC group, the knockdown efficiency of different concentrations of oligonucleotides was calculated, and the results are shown in Table 7. Taking SG21-40 as a control, among the 15 MsPA modified antisense oligonucleotides, 10 had better knockdown effect than SG21-40.

[0121] Table 7

[0122] No. Knockdown efficiency (%) No. Knockdown efficiency (%) Untreated -1 SG-346 -16.11 SG21-40 55.45 SG-347 71.34 SG-339 72.53 SG-348 94 SG-340 83.4 SG-349 41.9 SG-341 71.55 SG-350 10.94 SG-342 65.73 SG-351 72.74 SG-343 84.67 SG-352 77.89 SG-344 87.91 SG-353 57.32 SG-345 -87.23

[0123] Example 7: Inhibition of angiogenesis in vitro by MsPA modified antisense oligonucleotides after free uptake into umbilical vein endothelial cells (HUVEC)

[0124] The cells used in this example were human umbilical vein endothelial cells (HUVEC) (Pronova, CP-H082). In a 96-well plate, 50 μL of pre-cooled Matrigel was added to each well to avoid air bubbles, and the plate was placed in a 37°C incubator for 30 minutes. The HUVEC cells were digested and counted, and 1.5 x 10 4 After mixing Lipofectamine RNAiMax transfection reagent with different sequence oligonucleotides, the HUVEC cells were added to the mixture to make the final concentration of oligonucleotides 100 nM, and then the mixture was added to the 96-well plate with Matrigel. After 18-24 hours, 1 ml of 1 mM calcein AM was added to 50 ml of serum-free medium to make the final concentration 20 nM, and after incubation at room temperature for 30 minutes in the dark, the cells were washed with PBS for 2-3 times, and then immunofluorescence imaging was performed using 485 nm / 529 nm. AngioTool software was used to analyze and count the pictures. The results are shown in Table 8. Figure 5 and Figure 6As shown in Table 8, the average vessels length (Average Vessels Length) and the junction density were detected respectively, and compared with SG21-40, 14 of the 15 MsPA modified antisense oligonucleotides significantly reduced the average vessels length, and all the MsPA modified antisense oligonucleotides significantly reduced the junction density.

[0125] Table 8

[0126] No. Average blood vessel length (%) Vessel node density (%) Con. 100.00 100.00 SG21-40 35.38 99.25 SG-339 30.02 77.33 SG-340 23.75 62.93 SG-341 16.83 61.62 SG-342 42.87 90.47 SG-343 20.39 60.41 SG-344 23.57 77.33 SG-345 11.51 38.50 SG-346 9.72 40.15 SG-347 12.73 46.42 SG-348 26.70 74.41 SG-349 21.23 82.03 SG-350 18.25 61.24 SG-351 28.78 83.56 SG-352 17.99 57.66 SG-353 25.61 83.94

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not limited thereto; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that the technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. An antisense oligonucleotide targeting APLN, characterized in that, The nucleotide sequence of the antisense oligonucleotide comprises a sequence that differs by no more than 1, 2, 3, 4, 5, 6, or 7 nucleotides from any one of the sequences set forth in SEQ ID NO. 1-63.

2. The antisense oligonucleotide of claim 1, wherein, The antisense oligonucleotide contains at least one modified nucleotide; Optionally, the modified nucleotide includes, but is not limited to, at least one of 2'-deoxy-modified nucleotide, 2'-O-methoxyethyl-modified nucleotide, 5'-methyl-modified nucleotide, 2'-O-methyl-modified nucleotide, 2'-fluoro-modified nucleotide, locked nucleotide, non-locked nucleotide, conformationally restricted nucleotide, constrained ethyl nucleotide, abasic nucleotide, 2'-amino-modified nucleotide, 2'-O-allyl-modified nucleotide, 2'-C-alkyl-modified nucleotide, 2'-hydroxyl-modified nucleotide, 2'-O-alkyl-modified nucleotide, morpholino nucleotide, bridged nucleic acid, peptide nucleic acid, locked nucleic acid, non-locked nucleic acid, and 2'-(S)-constrained ethyl modified nucleotide.

3. The antisense oligonucleotide of claim 1, wherein, At least one of the internucleoside linkages is a modified internucleoside linkage; Optionally, the modified internucleoside linkage includes at least one of phosphorothioate internucleoside linkage and methylphosphonate internucleoside linkage; Optionally, all of the internucleoside linkages of the antisense oligonucleotide are phosphorothioate internucleoside linkages. Optionally, the antisense oligonucleotide contains 1, 2, or 3 methylphosphonate internucleoside linkages, and the remaining internucleoside linkages are phosphorothioate internucleoside linkages.

4. The antisense oligonucleotide according to any one of claims 1 to 3, characterized in that, The antisense oligonucleotide is a gapmer; Optionally, the antisense oligonucleotide comprises a gap segment consisting of at least 5, 6, 7, 8, 9, or 10 contiguous 2'-deoxy-modified nucleotides, and a 5' wing segment and a 3' wing segment each consisting of contiguous nucleotides, wherein the gap is located between the 5' wing segment and the 3' wing segment; Optionally, the nucleotides in the 5' wing segment and the 3' wing segment are each a modified nucleotide; Optionally, each modified nucleotide in the 5' wing segment and the 3' wing segment is independently selected from 2'-O-methoxyethyl-modified nucleotide, 2'-methoxy-modified nucleotide, locked nucleic acid, or bridged nucleic acid; Optionally, the nucleotides in the 5' wing segment and the 3' wing segment are each a 2'-O-methoxyethyl-modified nucleotide; Optionally, the cytosine nucleotides in the antisense oligonucleotide are each a 5'-methylcytosine nucleotide.

5. The antisense oligonucleotide of claim 4, wherein The antisense oligonucleotide comprises a gap segment consisting of 10 contiguous 2'-deoxy-modified nucleotides, and a 5' wing segment and a 3' wing segment each consisting of 5 contiguous nucleotides, the nucleotides in the 5' wing segment and the 3' wing segment are each a 2'-O-methoxyethyl-modified nucleotide, and the cytosine nucleotides in the antisense oligonucleotide are each a 5'-methylcytosine nucleotide; The antisense oligonucleotide contains 2 or 3 contiguous methylphosphonate internucleoside linkages, the methylphosphonate internucleoside linkages are located in the gap segment, and the remaining internucleoside linkages are phosphorothioate internucleoside linkages.

6. The antisense oligonucleotide of claim 5, wherein The nucleotide sequence of the antisense oligonucleotide is set forth in SEQ ID NO.

35.

7. A composite, characterized in that, The complex comprises: (a) the antisense oligonucleotide or salt thereof according to any one of claims 1-6; and (b) one or more delivery vehicles linked to (a).

8. Use of the APLN-targeting antisense oligonucleotide or salt thereof according to any one of claims 1-6, or the complex according to claim 7, in any one of: (I) reducing APLN mRNA levels in a subject; (II) manufacture of a medicament for reducing APLN mRNA levels in a subject; (III) reducing vessel length and / or vessel node density in a subject; (IV) manufacture of a medicament for reducing vessel length and / or vessel node density in a subject; (V) manufacture of a medicament for treating, preventing and / or alleviating a pathological condition or disease caused by APLN in a subject; (VI) treating, preventing and / or alleviating a pathological condition or disease caused by APLN in a subject. Optionally, the pathological condition or disease caused by APLN in a subject comprises, but is not limited to, at least one of hypertension, pulmonary arterial hypertension, atherosclerosis, peripheral arterial disease, glioma, lung cancer, hepatocellular carcinoma, portal hypertension, type 2 diabetes, diabetic vascular complications, obesity, retinopathy of prematurity, diabetic retinopathy, central retinal vein occlusion, age-related macular degeneration, and irreversible vision loss.

9. A pharmaceutical composition, characterized by, The APLN-targeting antisense oligonucleotide or salt thereof according to any one of claims 1-6, or the complex according to claim 7.

10. A method of inhibiting APLN in a subject, characterized in that, comprising contacting a subject with the APLN-targeting antisense oligonucleotide or salt thereof according to any one of claims 1-6, or the complex according to claim 7, or the pharmaceutical composition according to claim 9.

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